Page 209 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

676 Liquid Chromatography

steel columns are generally 5–30 cm in length with a able to design HPLC instruments particularly suited for
2.3–6.2-mm inside diameter. The length of the column separations of biological samples.
is often determined by the particle size of the packing
since pressure drop is inversely proportional to the square E. Column Packing Supports
of the particle diameter (dp). The pressure drop ( p) of
a packed bed can be more completely described as Column packing supports for HPLC can be divided into
two main classes, silica and polymer types. Silica (Fig. 5a)
2
p = ηL µφ/dp , has several desirable properties such as excellent pressure
where η = mobile phase viscosity, L = column length, stability, high surface area, good control of size and shape,
µ = linear velocity, and φ = flow resistance factor (≈500). and ease of availability. Of the silica packings, pellicular
Small diameter columns can have better peak sensitivity and porous are the two major kinds. Pellicular packings,
(see Section I.E) but sacrifice sample capacity. developed first as an alternative to classical large particle
Stainless steel zero dead volume fittings such as a LC supports, basically consist of a solid glass bead on
1 1 which a 1–2 µm porous layer of silica has been deposited.
16 in. → 16 in. union (Fig. 4a) are often required to make
connections. The 1 in. → 1 in. reducing union fittings Interaction of the sample components occurs only in this
4 16
(Fig. 4b) are used to connect the column to the injector and thin layer. Originally 40-µm size particles were made
detector. Both types of fittings can easily withstand 6000 but now smaller particles are available. Pellicular parti-
psi pressure. Pressed in the column fittings are 0.2-µm cles, fairly inexpensive in price, are sometimes used in
frits to contain the packing particles in the tube and prevent guard columns. Guard columns, usually 3–5 cm in length,
any particulates from reaching and disturbing the packing protect the analytical column from irreversibly adsorbed
bed. The connecting volume between the column-injector sampleconstituentsoftenfoundincomplexbiologicalma-
and column-detector must be kept at a minimum because trices.Inaddition,asilicaguardcolumncanprotectasilica
2
bandbroadening (variance or σ ) is directly proportional analytical column from dissolution due to a high pH mo-
to the fourth power of the tubing radius but only propor- bile phase by saturating the mobile phase with dissolved
tional to the column length, and inversely proportional to silica. Porous silica particles that are 10, 5, or 3 µmin
the flowrate. Therefore, 1 in. stainless steel tubing with size are more commonly packed in analytical columns.
16
a 0.01 in. or smaller inside diameter is used for all fitting Recently, columns packed with 2-µm particles have been
connections. Recently, metal free column hardware and reported in the literature. The 3–10-µm size particles can
polyethyletherketone (PEEK) tubing have become avail- be commercially obtained either irregular or spherical in
2
shape with a surface area ranging from 50 to 500 m /g.

1
1
FIGURE 4 HPLC zero dead volume 16 in. → 16 in. connect-
ing (A) and 1 in. → 1 in. reducing (B) unions. [From S. Schram
4 16
(1980). “The LDC Basic Book on Liquid Chromatography,” Milton
Roy Co., p. 79. Reprinted with permission from Milton Roy, Inc.,
and R. W. Yost, L. S. Ettre, and R. D. Conlon (1980). “Practi-
cal Liquid Chromatography,” Perkin–Elmer Corporation, Norwalk, FIGURE 5 Chemical structures of silica (a) and poly-
Connecticut, p. 245.] styrenedivinylbenzene (PSDVB) (b).